Assessing Introgression between European Wildcats (Felis silvestris silvestris) and Domestic Cats (Felis silvestris catus)

Abstract

Introgression is an important issue in evolutionarybiology. It is defined as the flow of genes between taxa through hybridization beyond the first generation. Introduced genes of a closely related taxon may serve as raw material for rapid adaptive evolutionary change. On the other hand, introgression could lead to reduced fitness in hybrids, i.e. outbreeding depression, if the newly mixed traits are maladapted to the environment or if, on the genomiclevel, co-adapted gene complexes are disrupted. In conservation biology, introgression is often seen as a threat to genetic integrity by genome swamping and as especially relevant when hybridization is human-induced. This is the case for the European Wildcat (Felis silvestris silvestris), which is hybridizing with the domestic cat (Felis silvestris catus), potentially since over 2500 years. Hence, assessing the impact of introgression by monitoring free-ranging cat populations is crucial for wildcat conservation. However, introgression is difficult to detect. In wildcats, neither morphological nor genetic methods allowed accurate recognition of introgression so far. In the present thesis, I aimed to provide the basic knowledge necessary to investigate the effects of introgression on wildcats by 1) developing genetic markers able to disclose introgression; 2) establishing a genotyping method for non-invasive samples that allow monitoring a wildcat population based on hair samples; 3) assessing the introgression rate in the wildcat population of the Jura region; and 4) describing hybridization patterns inwildcats from France, Germany and Switzerland. Chapter 1describes the development of single nucleotide polymorphism (SNP) markers, which allow reliable recognition of individual levels of introgression in wildcats, domestic cats and their admixed progeny. I first defined reference wildcats and domestic cats, based on the analysis of microsatellites, mitochondrial and Y-chromosome sequences, as well as morphological criteria. Secondly, I sequenced a selected small part of the genome of six reference wildcats and three domestic cats by high-throughput-sequencing. The comparison of the sequences revealed over 800’000 SNPs between both subspecies. I then selected 200 SNPs at which wildcats and domestic cats had differently fixed alleles and sequenced the regions on the genome containing these SNPs in an additional ten wildcats and 13 domestic cats, to validate the diagnostic value of these 200 SNPs. Using a Bayesian approach, I finally assessed the power of the 48 most differentiated SNP markers in determining the individual hybridization level ofsimulated hybrids until second generation of hybridization. This subset of SNPs allowed assessing the correct hybrid status with high accuracy, since 99.6% of the simulated individuals were assigned to the correct hybrid category. These SNP markers thus allow the reliable assessment ofintrogression levels in natural populations. Non-invasive sampling is a common and efficient way to sample elusive populations like wildcats. But the limited quality and quantity of nuclear DNA extracted from non-invasively collected samples, like single hairs, is a challenge for accurate genotyping. Chapter 2shows how I optimized a SNP genotyping method to yield reliable genotypes of single hairs. I developed a 96.96 Fluidigm SNP genotyping array (SNP chip), based on the nuclear diagnostic SNPs described in chapter 1 and on published mitochondrial (mtDNA) SNPs. The SNP chip contained 75 nuclear SNP markers most differentiated between wildcats and domestic cats for recognition of the introgression level, 11 nuclear Summary 9 markers and four mtDNA markers for recognition ofindividuals, four diagnostic mtDNA markers for maternal lineage assessment and two Y-linked markers for paternal lineage assessment and sex determination. Prior to genotyping, DNA extracted from single hairs was quantified with a cat specific real-time PCR assay. This step allows excluding hairs from species other than Felis silvestrisand hairs of too low DNA quantity and quality for furthergenotyping. To estimate the accuracy of these newly designed Fluidigm genotyping assays, I compared genotypes of 17 cats called with both Sanger sequencing and Fluidigm. Genotyping error was 0.9%. To estimate the accuracy of the genotyping method optimized for hairs, I comparedthe genotypes generated from both tissue and single hair samples of selected individuals. Genotyping error was 1.6%. These low error rates allowed correct recognition of individuals and assessment ofintrogression levels. This optimized genotyping method thus allows monitoring introgression rate in natural populations based on non-invasive hair sampling. In chapter 3, this optimized genotyping method was then applied to non-invasively and systematically collected hair samples of the cat population of the Swiss Jura, to assess its rate of introgression. Twenty one percent of the sampled wildcats wereintrogressed, based on the nuclear diagnostic markers. This corresponds to a migration rate from domestic cats to wildcats of 0.02 migrants per generation. In contrast, migration rate from wildcats into domestic cats was negligible, suggesting a directional introgression. Haphazard sampling of the same wildcat population, mostly via road kills, led to similar results. Hybridization was found to occur between wildcat male and domestic cat female as well as vice versa and, based on the occurrence of backcrosses, both female and male F1-hybrids seemed viable and fertile. The hybridization patterns observed in chapter 3 were confirmed in chapter 4, where I estimated introgression rates in a large set of free-ranging wildcats of France, Switzerland and Germany. I found 53 hybrids (11%) out of 491 samples, corresponding toa migration rate from domestic cat to wildcat of 0.02 migrants per generation. Migration rate fromwildcat into domestic cat was lower. Maternally inherited markers were more often introgressed thanpaternally inherited ones. Furthermore, hybrids seemed to concentrate at wildcat distribution edges. In addition, I found some evidence that the wildcat population of the Franco-Swiss Jura is possibly expanding. These results are all congruent with a selectively neutral model, where introgression could be seen as a mechanism of dispersal. Although the main findings of this thesis suggests that introgression might simply be a byproduct of wildcat range expansion, it would be an overhasty conclusion to state that introgression is not a risk to wildcats, since many important aspects, e.g. demography, ecology and time, were not sufficiently considered so far. Based on the precautionary principle, introgression should still be considered as relevant to species conservation. A key conservation goal in respect to the potential threat of introgression should be the knowledge of the mechanisms leading to introgression. Thus, one of the main conservation measures should be to monitor introgression in wildcat populations over time.